<p> The ansamycin antibiotics are unique antibacterial agents that inhibit bacterial DNAdependent RNA polymerase II. Clinical use of this class of antibiotics has primarily been focused on the treatment oftuberculosis using the semi-synthetic rifamycin derivative, rifampin. As drug resistance among different classes of antibiotics continues to rise, there is increased interest in new applications ofrifamycins for diseases other than tuberculosis. Clinical resistance to rifampin has largely been the result of point mutations in the target, RpoB, however chromosomal and transposon mediated enzyme-associated resistance is well documented. As rifamycin antibiotic use becomes more widespread, enzymatic resistance will inevitably become more prevalent. Here we describe the characterization of one of the principle enzymes associated with rifampin inactivation, the rifampin ADP-ribosyl transferase enzyme (ARR). Two chromosomally encoded ARR enzymes from MYcobacterium smegmatis, and Streptomyces coelicolor, and the Tn-encoded ARR-2, widely distributed in Gram negative pathogens, were overexpressed and characterized. These enzymes exhibit comparable, substrate specific steady state kinetic features, and substrate-induced conformational changes that suggest ARR enzymes may demonstrate a preferred order of substrate binding. To gain further insight into the interaction between ARR enzymes and rifampin and NAD+, the three-dimensional crystal structure of ARR from M smegmatis was solved in complex with rifampin. Based on the threedimensional structure of ARRm, an SNl type reaction has been predicted for rifampin ADPribosyl transferase enzymes. This is the first detailed examination of these novel antibioticmodifying enzymes, relevant to their increased use in the clinic. </p> / Thesis / Master of Science (MSc)